1. Field of the Invention
The invention relates to the so-called "electro spray ionization" of molecules (frequently abbreviated to ES or ESI). This method is, in general, carried out by spraying aqueous solutions from a spray capillary under the influence of an electric field. The method is especially suited for the ionization of large molecules, especially bio molecules, and for their analysis by mass spectrometry after introduction into the vacuum system of the spectrometer.
2. Description of the Related Art
The conventional method of electrospray ionization, as recommended for commercially available instrumentation, operates in the following way.
A voltage of several kilovolts is applied between a metal capillary and a flush surface separated by a distance of approximately 20 to 50 millimeters. Under the effect of the electric field, a liquid in the capillary is dielectrically polarized at the end of the capillary and pulled out into a cone, the so-called Taylor cone. At the pointed end of the cone the surface tension of the liquid is no longer able to withstand the attraction of the electrical field, causing a small droplet, which is electrically charged due to dielectric polarization, to be detached. Under the effect of the inhomogeneous electrical field, the charged droplet flies greatly accelerated towards the flush counterelectrode, but is soon slowed down in the surrounding air. During the flight considerable evaporation occurs from the surface of the droplet. Should the liquid contain a few larger molecules, which can be charged (ionized) more easily than the molecules of the liquid, the larger molecules remain in ionized form after complete evaporation of the liquid. Under the effect of the electrical field, the ionized molecules fly on towards the counterelectrode due to the known process of "ion mobility" and can be transferred to the vacuum system of a mass spectrometer through a fine aperture or through a capillary.
Depending on the supply of liquid in the capillary, the droplets detach with great frequency, generally resulting in a continuous stream of charged particles or ions. The supply of liquid is maintained by a very uniformly operating pump, usually a syringe pump. Other systems use the electroosmotic pumping effect of capillary electrophoresis.
In this process, larger molecules are usually charged not only singly, but a number of times. As a rough rule, the average charge number increases in accordance with the size of the molecule. For the most part the charge is not ionization by electron abstraction, but by protonation, i.e. combination with charged hydrogen atoms H.sup.+. For this reason, ionization also depends greatly on the hydrogen-ion concentration, i.e. on the pH value of the solution. There is usually a wide distribution around the average charge number with different numbers of charges.
The multiple charge of the large molecules and the wide charge distribution are particularly favourable for detection. Since most mass spectrometers have a limited mass range (or more precisely a limited range of mass-to-charge ratios), it is still possible, despite this limitation, to detect very large molecules well beyond the mass range defined for singly charged ions since the electro spray ions are charged a number of times. Due to the wide and regular distribution of the number of charges over molecular ions of the same isotopic mass pattern, it is easily possible to determine the molecular mass.
U.S. Pat. Nos. 5,115,131 and 4,531,056 describe ESI using a capillary, and are believed to represent the state of the art. These references and the other patents and literature articles cited in them are incorporated herein by reference. The closest state of the art concerned with electrospraying at ambient pressure is described in U.S. Pat. No. 4,531,056. U.S. Pat. No. 5,115,131 is concerned with electrospraying in a vacuum. Under such conditions a kind of explosive evaporation of the solution takes place at the tip of the capillary under influence of the surrounding vacuum.
All presently known techniques of electrospraying teach that it is necessary (a) to take active steps to cause the solution to flow through the capillary and (b) to spray the outflowing solution (the "eluent") by a high electric voltage. This is emphasised in the Claims of the patents referred to above. For example, the claims of U.S. Pat. No. 4,531,056 refer to causing the solution to flow through the capillary as an independent step.
U.S. Pat. No. 5,115,131 especially teaches that it is possible to operate, using external pumping of the solution, with extremely low flow rates in the range of 0.1 to 100 nanoliters per second (or 1 to 10 nanoliters per second in a narrower rage) by using capillaries with very fine tips. The patent does not teach how the ions can be introduced into the vacuum system of the mass spectrometer. Furthermore, the patent description is directed entirely to spraying in vacuum with its evaporation by the surrounding vacuum. There is no suggestion that it is possible to employ such low flow rates in ambient gas. Again, to "cause the solution to flow through the capillary" is an independent step in claim 1.
The flow in all these cases is generated by the operation of external pumps, most often a syringe pump, or an HPLC pump in case of coupling with HPLC. U.S. Pat. No. 4,885,076 describes the production of a flow by electroosmotic pumping in an electrophoresis capillary.
There are significant disadvantages in the existing prior art. Frequently in biochemistry and medicine only very small substance quantities are available for analyses (sometimes quantities of only a few femtomoles). Assuming however, a lower concentration limit of approximately 0.1 picomol per microliter and a minimum flow rate of one microliter per minute, the lower limit for the consumption of analyte is, however, approximately 100 femtomol per minute. Scanning, especially with the MS/MS method, usually takes several minutes. Several hundred femtomol of substance are therefore required. In routine methods these values rise by factors of 10 to 100, meaning that quantities of well over one picomol are regularly required.
Particularly useful results can be obtained by "tandem mass spectrometry" techniques (commonly abbreviated as "MS/MS"), in which ions of a preselected mass-to-charge ratio are filtered and fed to a fragmentation cell. In the fragmentation cell, these preselected "parent" ions are dissociated into neutral fragments and "daughter ions". The daughter ions are measured in a second mass spectrometric method. MS/MS techniques can be carried out as "tandem in time", in which, in a first time period, ions of a preselected mass-to-charge ratio will be "isolated" by the ejection of all other ions. The isolated ions can then be fragmented in a second time period, for example by exciting their secular oscillations in the presence of a collision gas. The daughter ions can then measured in a third time period, resulting in a daughter ions spectrum of the parent ions.
Attempts to reduce the flow rate for electrospraying in ambient air at standard pressure, which with conventional and present commercially available instruments is at least approximately 1 microliter per minute, have failed so far since they did not achieve stable electrospray conditions. With the conventional method the flow rate is always determined by a pump. It is very difficult or impossible to stabilise the pumping process through the network of lines and connections at such extremely low flow rates.
Another disadvantage is the production of rather large droplets which need a long flight path for evaporation. The droplets have diameters of about 1 micrometer.
One improvement of this method which has been suggested involves surrounding the solution with a second liquid having a different surface tension using the aid of a coaxial capillary in order to thus achieve smaller droplets (U.S. Pat. No. 5,122,670). The coaxial feeding of a special additive spray gas to pneumatically assist spraying has also been proposed (U.S. Pat. No. 4,861,988; U.S. Pat. No. 5,170,053).
A further improvement which has been proposed is to assist the spraying process using ultrasound. This also results in somewhat smaller droplets.
It is, however, known to all specialists in the field that analyte is positively wasted in the conventional method of electro spraying. The long distance required for evaporation of the droplets necessarily results in considerable widening of the ion beam due to space charge repulsion so that only a small proportion of the ions (approximately 1/100 to 1/1,000) can be admitted through the fine aperture of the mass spectrometer.
Present electrospraying can be influenced by very many parameters of the solution used, most parameters having very narrow tolerance limits, outside of which stable operation is not possible. For example, highly pure water cannot be sprayed since its electrical resistance is too great. On the other hand, the addition of salts to increase conductivity is exceptionally critical. Normally, salt concentrations of less than one millimole per liter are employed. Salt solutions of over 10 millimole per liter cannot be used.
Aqueous solutions with high proportions of organic solvents, such as result from separation processes with liquid chromatography or gel chromatography, can also not be sprayed with the conventional method.
The range of pH values of the solution is also greatly limited, primarily to the slightly acid range. Owing to this limitation in pH, negative spraying, as is required for nucleotides, is not possible at all.
It is therefore an object of this invention to provide a method of electrospraying using extremely small amounts of the substances to be ionised (the analyte) and operating in a stable manner at wide ranges of solution viscosities, pH values, salt concentrations, and concentration of organic solvents. The spray method should be applicable to the production of positive and of negative ions.